Technical Notes

4 TECHNICAL NOTE ©2019 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. 0 1 8 . 6 3 S i g n a l Q u a l i t y 1 5 0 m M N a C l K C I M g C I 2 C a C l 2 G S H T C E P E D T A G l y c . P E G 3 0 0 3 0 0 0 50 1 5 0 1 5 0 50 0 T R I S H E P E S P P T R I S 1 0 H E P E S 1 0 1 0 5 0 T w P l N a C l i n m M P l u r o n i c ® F - 1 2 7 T w e e n ® 2 0 D M S O 2 . 5 % D M S O 2 . 5 % D M S O 5 % D M S O 5 % P P 1 0 1 0 5 0 5 0 1 0 T w P l T w P l A F E D C B G H 1 2 8 7 6 5 4 3 12 11 10 9 Buffer condition A1 T: Target only T+L: Target ligand complex T T+L T T+L Figure 2: Le panel: pipetting scheme for the 384-well Dianthus assay plate. All odd columns were filled with target only (reference, green), all even columns with the target-ligand mix (positive control, purple). For this buffer screen, each buffer formulation from the Buffer Exploration Kit is used in four wells of the 384-well assay plate. Right panel: resulting heatmap showing the SQ for all 96 different buffer conditions, calculated from duplicates of reference and positive control. Each square in the heatmap corresponds to the four wells where the corresponding buffer formulation was measured. The SQ value is represented as color intensity. impact of a buffer component can be identified by clustered SQ values and their corresponding colors in the heatmap. The color pattern shown in Figure 2 (right panel) indicates that in conditions with 10 mM CaCl 2 (C7-C10) the SQ is higher than in all other conditions. In direct comparison to almost identical buffer conditions without CaCl 2 (compare C7 with C1, C8 with G1, C9 with C3 and C10 with G3) it is apparent that the addition of 10 mM CaCl 2 results in an increase in SQ for HEPES buffer and even larger increase for TRIS buffer. In HEPES buffer, the addition of MgCl 2 also improved the SQ (B9 and B10). When comparing B9 and B10 to corresponding respective results from wells C3 and G3, which are the same buffer conditions without MgCl 2 , the SQ was enhanced. Figure 3 shows the results obtained for four selected buffers in more detail, representing exemplary good and bad assay conditions. With an SQ of 6.54, the buffer condition in square C3 (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005 % Tween®20) shows good applicability as an assay buffer. However, by adding 10 mM CaCl 2 SQ can be increased more than two-fold, as shown in square C9 (SQ = 18.63). In contrast, when comparing the SQ obtained for squares C3 and F9, it is clear that the addition of 5 mM EDTA results in a drop in the SQ to 1.09 as no binding of SAM to G9a is detectable under these conditions. The addition of 1 mM TCEP is also disadvantageous, as shown in the SQ for E9. Furthermore, the aberrant TRIC trace obtained for one of the reference replicates in E9 (green trace) is a typical indication for protein aggregates in TRIC assays.

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